Non-Hodgkin lymphoma (NHL) is a prevalent cancer with an estimated 70,800 cases predicted to be diagnosed in the United States in 2014, with the incidence doubling since 1980. Although treatments for NHLs greatly improved following the FDA approval of rituximab, refractive malignancies still occur that are nonresponsive to current therapies in about half of all patients, indicating that improved treatment strategies are needed. One of the most reliable biomarkers and therapeutic targets for B cell NHL is CD20, which is a non-internalizing antigen that remains on the cell surface when bound to a complementary antibody. The crosslinking of CD20-bound antibodies with a secondary antibody results in apoptosis. The overall goal of this project is to generate new, drug-free macromolecular therapeutics for improved treatment of NHL. A new apoptosis induction system is proposed based on the biorecognition of two complementary morpholino oligonucleotides at the cell surface, crosslinking of CD20 antigens and initiation of apoptosis. The system is composed of a pair of complementary morpholino oligonucleotides; Fab' fragment of the 1F5 anti-CD20 antibody; and HPMA copolymer. One oligonucleotide (MORF) is conjugated to the Fab' fragment, the other (cMORF) is conjugated in multiple grafts to polyHPMA. Indeed, the exposure of CD20+ Raji B cells to Fab'- MORF conjugate resulted in the decoration of the cell surface with multiple copies of MORF via antigen- antibody fragment biorecognition. Further exposure of the MORF decorated cells to HPMA copolymer grafted with multiple copies of cMORF (P-(cMORF) x) resulted in oligonucleotide hybridization and formation of MORF-cMORF heterodimers on the cell surface. This second biorecognition event induced crosslinking of CD20 receptors and triggered apoptosis of B cells. The structure of nanoconjugates will be optimized based on the structure and length of oligonucleotide sequences; molecular weight of backbone degradable HPMA- based carrier; number of grafts per macromolecule; spacing of grafts along the macromolecule; use of flexible spacer separating the cMORF grafts and backbone and/or MORF and Fab'. The system will be validated based on data on five cell lines with varying degree of CD20 expression: Raji, Daudi, Ramos, Namalwa, and DG-75. The efficacy of drug-free macromolecular therapeutics will be evaluated on three NHL animal models using lymphoma cells with different phenotype: Raji, SUDHL4, and Z138. For imaging studies luciferase expressing Raji-luc cells will be used. Body distribution, impact of the dose and frequency of administration and time lag between the administrations of two nanoconjugates on the efficacy of treatment will be evaluated with the aim to select leading compound(s) and optimal treatment modalities. The optimized conjugates will be evaluated on cells isolated from patients with clinically common NHLs that express CD20: chronic lymphocytic leukemia, mantle cell lymphoma, diffuse large B cell lymphoma, and follicular lymphoma. This will permit to identify subsets of patients suitable for the new therapy.